Late Viral Protein Synthesis Research Articles

Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis

(35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization.

Changes in the Surface of Virus‐Infected Cells Recognized by Cytotoxic T Cells

P-815 mastocytoma cells developed susceptibility to immune T-cell-mediated cytolysis shortly after infection by ectromelia virus. Intracellular viral replication and late protein synthesis seem to bu unnecessary events. Interference with early protein synthesis, however, inhibits the development of susceptibility to lysis. The important intracellular events necessary for subsequent cytolysis appear to occur within 1 hour of infection. Virus rendered non-infectious by ultraviolet irradiation but not by gamma irradiation is able to induce these changes. By determining the minimum and essential events of the infectious process which result in T-cell-mediated cytolysis, the task of establishing the molecular changes occurring in the target cell surface membrane necessary for immune T-cell recognition should be simplified.

Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them

We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious virus yields declined progressively in passages 1 through 10 and gradually increased at passages 11 through 14. The yields correlated with PFU/particle ratios. (ii) In cells infected with virus from passages 6 through 10, there was an overproduction of an early viral polypeptide (no. 4) and a delay in the synthesis of late viral proteins. In addition, the virus in these passages interfered with the replication of a nondefective marker virus. Cells infected with passage 14 virus produced normal amounts of polypeptide 4 and, moreover, this virus showed minimal interfering capacity. (iii) In addition to DNA of density 1.726 g/cm-3, which was the sole component present in viral progeny of passage 0, passages 6 through 14 contained one additional species (p 1.732) and in some instances (passages 6 and 10) also DNA of an intermediate buoyant density. The ratio of p 1.732 to p 1.726 DNA increased to a maximum of 4 in passages 6 through 9 and gradually decreased to 1 in passages 10 through 14. (iv) p 1.732 DNA cannot be differentiated from p 1.726 DNA with respect to size; however, it has no Hin III restriction enzyme cleavage sites and yields only predominantly two kinds of fragments with molecular weights of 5.1 x 10-6 and 5.4 x 10-6 upon digestion with EcoRI enzyme. (v) Partial denaturation profiles of purified p 1.732 DNA from passage 14 revealed the presence of two types of tandemly repeated units corresponding roughly in size to the EcoRI fragments and situated in different molecules. (vi) In addition to the two kinds of p 1.732 molecules consisting of tandem repaeat units of different sizes, other evidence for the diversity of defective DNA molecules emerged from comparisons of specific infectivity and interfering capacity of the progeny from various passages. The data suggest that some of the particles with DNA of normal buoyant density (1.726) must also be defective since the capacity to interfere and to produce an excess of polypeptide 4 did not appear to be proportional to the amount of high-buoyant-density defective DNA. The data suggest that defective interfering particles are replaced by defective particles with diminished capacity to interfere and that more than one species of defective DNA molecules evolves on serial preparation of HSV.

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."

Anti-poxvirus activity of rifampicin associated with hydrazone side chain.

RIFAMPICIN1 inhibits the replication of vaccinia virus in infected cells in vitro when added at 100 µg/ml. immediately after infection3,4 or at a late stage of the virus latent period4. In the presence of the drug the translation of late viral mRNA spacies was found to be markedly inhibited 8 h after infection5; the rate of late viral protein synthesis declined6; the synthesis of the particulate viral RNA polymerase molecules, induced late in infection, was prevented7; and the assembly of virions was affected8,9. Rifampicin did not affect the synthesis of viral mRNA molecules nor their association with ribosomos to form polyribosomes5. The inability of rifampicin to inhibit in vitro the activity of the three different species of poxvirus specific DNA dependent RNA polymerases7 suggested that the mode of antiviral activity of rifampicin differed from its antimicrobial activity. The latter was attributed10 to the macrocyclic ring of the rifamycin SV molecule which binds to the microbial DNA dependent RNA polymerase and inhibits the initiation of RNA synthesis. Using Shope fibroma virus (SFV), a poxvirus which causes localized tumours in rabbits11–13, and several rifamycin SV derivatives, we have obtained results which indicate that the anti-poxvirus activity resides in the hydrazone side chain of rifampicin.

Biochemical studies on adenovirus multiplication. XVI. Transcription of the adenovirus genome during abortive infection of elevated temperatures.

The synthesis of infectious virus is reduced by 99% in adenovirus type 2-infected KB cells maintained at 42 C in suspension. Studies to delineate the steps in virus biosynthesis blocked at 42 C revealed the following. (i) The inhibition of ribosomal ribonucleic acid (RNA) synthesis and the block in the conversion of ribosomal RNA precursors to ribosomal RNA which normally occur in infected cells at 37 C was accentuated at 42 C. (ii) The same species of early and late viral messenger RNA were synthesized at 37 and 42 C. (iii) The shift from host cell deoxyribonucleic acid (DNA) synthesis to viral DNA replication occurred earlier at 42 C than at 37 C. These findings indicate that the thermosensitive block(s) in virion formation does not occur at the level of viral DNA replication or transcription of viral mRNA but probably involves the synthesis of late viral proteins or the maturation of the virion.

Vaccinia virus directed RNA and protein synthesis in the presence of rifampicin

The effects of rifampicin, an inhibitor of bacterial DNA transcription, on the formation of infectious vaccinia virus and viral particles, RNA and proteins were studied under single step growth conditions. Although virion formation was inhibited by more than 99%, the synthesis of early and late viral directed RNA species, as characterized by their sedimentation properties, was unaffected. Rifampicin did not prevent the synthesis of early and late viral proteins identified by immunodiffusion, disc gel electrophoresis and disc gel immunoelectrophoresis. The drug also did not inhibit the in - vitro activity of the virion associated RNA polymerase.